Identification of collaborative cross mouse strains permissive to Salmonella enterica serovar Typhi infection

Salmonella enterica serovar Typhi is the causative agent of typhoid fever restricted to humans and does not replicate in commonly used inbred mice. Genetic variation in humans is far greater and more complex than that in a single inbred strain of mice. The Collaborative Cross (CC) is a large panel of recombinant inbred strains which has a wider range of genetic diversity than laboratory inbred mouse strains. We found that the CC003/Unc and CC053/Unc strains are permissive to intraperitoneal but not oral route of S. Typhi infection and show histopathological changes characteristic of human typhoid. These CC strains are immunocompetent, and immunization induces antigen-specific responses that can kill S. Typhi in vitro and control S. Typhi in vivo. Our results indicate that CC003/Unc and CC053/Unc strains can help identify the genetic basis for typhoid susceptibility, S. Typhi virulence mechanism(s) in vivo, and serve as a preclinical mammalian model system to identify effective vaccines and therapeutics strategies.


Results
CC mice are permissive to S. Typhi replication in vivo. Since S. Typhi is a human-restricted pathogen, researchers often employ S. Typhimurium, a closely related bacterium that causes "typhoid-like" disease in mice. Organs of the mononuclear phagocytic system, such as spleen and liver, are the major sites of replication of S. Typhi in humans and this pattern is well-reflected in the i.p. route of infection of S. Typhimurium in mice 34,35 . To test whether CC mice are permissive or not, we infected 9 different CC mouse strains, 4 CC progenitors and BALB/cJ of both sexes i.p. with 2 × 10 4 CFUs of S. Typhi strain Ty2. We assessed bacterial burden in the spleen 6 days post-infection to allow time for a detectable S. Typhi replication or host clearance. BALB/cJ, all 4 CC progenitor strains (C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, and CAST/EiJ), and several CC strains (CC035/Unc, CC030/GeniUnc, CC022/GeniUnc, and CC052/GeniUnc) showed little-to-no bacteria in the liver (Fig. 1). The bacterial count in the spleens of all these mice was lower than or close to the number of bacteria initially injected (Fig. 1). Some CC017/Unc females, CC038/GeniUnc and CC055/TauUnc mice showed a marginal increase in the bacterial burden in the liver and spleen (Fig. 1). Although an increase in bacterial load was not seen consistently in the livers of all CC003/Unc and CC053/Unc mice, the bacterial load in the spleens of these mice was significantly higher than that observed in the commonly used laboratory strains BALB/cJ, C57BL/6J, and 129S1/ SvImJ (Fig. 1), indicating bacterial growth in these two CC strains. Interestingly, we observed a significant difference in the bacterial burden between male and female CC003/Unc mice (Fig. 1), a pattern not observed in the spleens of CC053/Unc mice (Fig. 1). These data suggest that CC053/Unc males and females, and CC003/Unc males but not females are permissive to S. Typhi replication in vivo.
Histological features of S. Typhi infection in the livers of CC mice recapitulates that of human and murine typhoid pathology. Since preliminary screening of CC strains suggested the permissiveness to S. Typhi infection in CC003/Unc and CC053/Unc mice (Fig. 1), we examined hematoxylin and eosin-stained liver specimens of these two CC strains to characterize histopathology. We found that the livers of CC003/ Unc mice had normal tissue architecture ( Fig. 2A,B). However, upon S. Typhi infection the liver tissue showed signs of steatosis, an abnormal retention of lipids within hepatocytes (Fig. 2D), as compared to uninfected mice (Fig. 2C). This indicates a perturbation of liver metabolism in infected mice. Furthermore, S. Typhi-infected CC003/Unc and CC053/Unc mouse livers exhibited lesions that consist of mononuclear cell infiltrates, congestion of sinusoids, and altered staining with little/no steatosis in hepatocytes proximal to the lesion (Fig. 2E-H). While tissue biopsies are rare in typhoid-infected patients, biopsies have been collected in cases where typhoid is not initially considered and are used to aid diagnosis 36 . These rare cases provide some of the only information known about the histopathology of typhoid. Interestingly, the lesions observed in CC003/Unc and CC053/Unc www.nature.com/scientificreports/ mice ( Fig. 2E-H) appear very similar to those described for human typhoid patients 37 . As a comparison, we also show that S. Typhimurium infection results in similar liver pathology that is commonly observed in inbred mice, C57BL/6J (Fig. 2I,J) and 129S1/SvImJ (Fig. 2K,L).
CC003/Unc and CC053/Unc mice contain all the major subsets of cells of the innate and adaptive immune system. Susceptibility of mice to an infection can be due to immune deficiency. Since immune competency of CC mice is necessary for studying protective responses to S. Typhi in preclinical models, we performed a comprehensive flow cytometric analysis of cells in the spleen and coelomic cavity of CC003/ www.nature.com/scientificreports/ Unc and CC053/Unc mice, as well as C57BL/6J mice as a control. In mice, the mature B cells can be divided into 4 subsets, namely, Fo, MZ, B1a, and B1b cells. Each of these subsets occupy a distinct functional niche in protective immunity 38 . For example, antibody responses to ViPS in mice are generated primarily by B1b cells 39 .
We found that all 4 major B cell subsets including B1b are present in CC003/Unc and CC053/Unc mice and their frequency is within the range observed in C57BL/6J mice, except the frequency of MZ B cells, which is higher in the CC mice compared to C57BL/6J mice (Fig. 3A vs. B,C). The ratio of CD4 to CD8 T cells in C57BL/6J mice is typically 2-3:1 40 , and we found a slightly altered ratios in CC003/Unc and CC053/Unc mice (Fig. 3). It has been reported recently that there is a high variablility in the CD4 and CD8 ratios as well as total B cell frequencies in CC strains 41  CC003/Unc mice are immunocompetent. Among the 9 CC strains of both sexes screened, CC003/Unc male mice showed relatively high susceptibility to S. Typhi replication as determined by the bacterial load in the liver and spleen in more than 6 independent experiments comprising of a total of 43 mice (Fig. 1). To test the proof of principle that CC mice can generate protective immunity against S. Typhi, we chose the CC003/Unc strain to test vaccination efficacy. We immunized male CC003/Unc mice with either plain ViPS or heat-killed S. Typhi. We found that both types of immunizations induced a robust anti-ViPS IgM response that peaked at 7 days post-infection (Fig. 4IA,IIA) as in immunocompetent C57BL/6J mice 35 and the serum obtained from the CC003/Unc mice at this time point was capable of killing S. Typhi in vitro in a complement-dependent serum bactericidal assay (Fig. 4IC,IIC). IgM response typically declines after 7 days post-infection, which is concurrent with the induction of IgG response due to antibody isotype switching 35 . Both immunizations generated an anti-ViPS IgG response by 21 days post-infection (Fig. 4IB,IIB), and serum obtained at this time point also killed S. Typhi in vitro (Fig. 4IC,IIC). These data suggest that CC003/Unc mice undergo isotype switching normally and the antibodies generated by these immunizations can provide protection. To test whether anti-ViPS antibodies confer protection in vivo, we challenged male CC003/Unc mice with S. Typhi strain Ty2 and measured bacterial load 6 days later as in Fig. 1. Although ViPS-immunized mice exhibited a reduced bacterial burden in the liver and spleen compared to unimmunized mice, the difference was not statistically significant ( Fig. 4ID,E), consistent with the low efficacy of the plain ViPS vaccine 7 . Immunization with whole bacteria induces a qualitatively different antibody response 42 . In a S. Typhimurium "surrogate" challenge model, we have previously shown that immunization with heat-killed S. Typhi controls bacterial burden better than ViPS immunization 35 . Here we found that immunization of CC003/Unc mice with heat-killed S. Typhi confers more efficient protection compared to ViPS, as determined by a significant decrease in bacterial burden in the liver and spleen (Fig. 4IID,E). Unlike adults, young children and infants do not respond to polysaccharide antigens such as ViPS. We have previously shown that 3-week-old mice (like young children) do not respond to ViPS efficiently due to a restricted antibody repertoire 43 . Therefore, we compared antibody responses to ViPS in young (3-week-old) and adult (8-12-week-old) CC003/Unc mice. We found that compared to adult CC003/Unc mice, the 3-week-old CC003/Unc mice are not capable of mounting an efficient IgM response to ViPS (Fig. 5A) and their serum was www.nature.com/scientificreports/ inefficient in killing S. Typhi in vitro (Fig. 5B). In summary, this data demonstrate that CC mice are not only immunocompetent but can control S. Typhi burden upon immunization and suggest that both young and adult CC003/Unc mice can serve as an experimental system to identify novel typhoid vaccines and therapeutics.

Discussion
One of the barriers to advancing the treatment and prevention of typhoid is the lack of a suitable animal model to study S. Typhi infection 8 . Since S. Typhi is strictly human adapted, S. Typhimurium, a natural pathogen of mice became the most widely used bacterium to understand pathogenesis and immunity in several inbred strains (e.g., C57BL6/J and 129/Sv). S. Typhimurium causes a "Typhoid-like" systemic disease in mice and shares 90% of genes with S. Typhi 44 . However, ~ 600 S. Typhi genes, including those encoding for ViPS biogenesis and typhoid toxin, are not found in S. Typhimurium 44 . Furthermore, several genes found in S. Typhimurium are pseudogenes in S. Typhi 45 . Therefore, wildtype S. Typhimurium strains cannot serve as a synonymous model system to decipher the role of S. Typhi-specific virulence factors. To understand the function of ViPS in vivo, Baumler and coworkers introduced all the S. Typhi genes required for ViPS biogenesis in S. Typhimurium 46 . Using this "chimeric" S. Typhimurium strain e.g. TH170 46 , a role for ViPS in resisting C3 deposition and complement receptor 3-mediated clearance 47 , in evading of TLR4 recognition 48,49 , and microbe-guided neutrophil chemotaxis was identified 50,51 .
The length of LPS of S. Typhimurium is very long compared to that of S. Typhi. In S. Typhimurium the length of LPS is controlled by the FepE gene product, which is a pseudogene in S. Typhi. To mimic the surface characteristics of S. Typhimurium to resemble that of S. Typhi, the S. Typhimurium strain TH170 was further engineered by deleting the FepE gene 4 . This strain of S. Typhimurium, referred to as RC60, was shown to exhibit cell surface and other characteristics of S. Typhi 4 . Using the S. Typhimurium strain RC60, we were able to identify several aspects of the anti-ViPS antibody repertoire required for protective immunity in vivo 35,43,52 . Thus, the use of S. Typhimurium as a "surrogate" model to understand certain virulence mechanisms of S. Typhi has been justified. However, the heterologous expression of S. Typhi genes in S. Typhimurium may not always permit identification of the role of certain S. Typhi-specific components. For example, the typhoid toxin of S. Typhi appears to be host-adapted and binds to N-acetylneuraminic acid (Neu5Ac) that is abundantly expressed in humans but  www.nature.com/scientificreports/ not in mice 53,54 . Therefore, studying the role of typhoid toxin in the pathogenesis requires a permissive mouse model that contains human tissue, such as "humanized" mice. Interestingly, a genome-wide analysis of S. Typhi mutants in "humanized" mice confirmed that ViPS is essential for virulence but surprisingly not the typhoid toxin 55 . The controlled human infection model also revealed that typhoid toxin is neither required for infection nor for the development of early typhoid fever symptoms 56 . Since the human challenge model represents early events of typhoid, the role of typhoid toxin in severe disease or the establishment of bacterial carriage remains to be determined. Host genetics clearly play a role in a variety of infectious diseases, as evidenced by hypothesis-driven analyses of polymorphisms in the Tlr4 gene 57 . In fact, CC mice were developed as a resource for mammalian systems genetics 25 . Few susceptibility genes e.g., Slc11a1 and Tlr11 have been implicated for the permissiveness of S. Typhi. Notably, the relative susceptibility or resistance to S. Typhimurium, a close relative of S. Typhi, was shown to depend upon a single nucleotide polymorphism in the Slc11a1 gene that encodes an ion transporter commonly referred to as Natural resistance-associated macrophage protein 1 (Nramp1) 58 and neutrophil cytosolic factor 2 (Ncf2) 59 . In the UNC systems genetics database, (https:// csbio. unc. edu/ CCsta tus/ index. py), we found that both CC003/Unc and CC053/Unc mice have the Slc11a1 resistant allele inherited from the progenitor A/J and the ncf2 allele is not mutated 60 . Although the role of Tlr11 is an ongoing controversy 18,19 , we have not found any mutation in the Tlr11 gene either in CC003/Unc or CC053/Unc mice. Therefore, a comprehensive genetic approach may help us discover novel genes responsible for susceptibility to S. Typhi in CC003/Unc and CC053/Unc. For example, one approach is to perform classical genetic crosses to generate F1 hybrids and F2 offspring by crossing either CC053/Unc or CC003/Unc with BALB/cJ, a known S. Typhi resistant and a non-CC progenitor strain, respectively. The susceptibility of F1 hybrids and F2 offspring to S. Typhi can be tested as in Fig. 1, and the genotype of those mice can be determined using the Giga Mouse Universal Genotyping Array (GigaMUGA) 61 or MiniMUGA 62 to find associations between resistance/susceptibility phenotype and genotype. Indeed, using a combination of genotyping and quantitative trait loci mapping, several S. Typhimurium infection susceptibility loci have recently www.nature.com/scientificreports/ been identified in CC042/GeniUnc mice 32 . Although CC042/GeniUnc mice inherited the susceptible Slc11a1 locus from the C57BL/6 progenitor and its Tlr4 gene product is functional, the bacterial burden in the CC042/ GeniUnc mice was 1000-fold more than that in C57BL/6N mice, suggesting that the hyper-susceptibility is due to other factors. Interestingly, CC042/GeniUnc mice also showed lower spleen weights and decreased B, T, and myeloid cell populations compared to control C57BL/6N mice, suggesting that an abnormality in the architecture of the immune system or a partial immune deficiency may be responsible for the hyper-susceptible phenotype. Indeed, an F2 cross between CC042/GeniUnc and C57BL/6N mice identified a susceptibility locus accompanied by a loss-of-function variant of the integrin alpha L (Itgal) gene 31 , which encodes for LFA-1, a molecule central to immune cell adhesion and trafficking. This hyper-susceptibility phenotype has been confirmed independently by comparing S. Typhimurium infection in C57BL/6N and LFA-1 −/− mice 31 . Unlike the CC042/GeniUnc mouse system for S. Typhimurium hyper-susceptibility, the reason for CC003/Unc and CC053/Unc mice susceptibility is unlikely to be associated with an altered architecture/composition of the immune system. The Itgal gene of CC003/Unc and CC053/Unc mice is inherited from C57BL/6J and 129S1/SvImJ progenitors, but not from WSB/ EiJ progenitor as in CC042/GeniUnc mice. Most importantly, CC003/Unc and CC053/Unc mice are immunocompetent (unlike CC042/GeniUnc or "humanized" mice) and possess all the major populations of innate and adaptive immune cells (Fig. 3) and respond to both T cell-independent immunogen (i.e., isolated plain ViPS) or T cell-dependent immunogen (i.e., heat-killed S. Typhi) (Fig. 4). Feaco-oral route is a natural mode of S. Typhi transmission in humans, that eventually results in a systemic infection. When we attempted oral infection using a gavage needle (10 9 CFU of strain Ty2 in 100 μl PBS), we did not observe permissiveness of S. Typhi infection in CC003/Unc mice. Therefore, we chose i.p. infection which is commonly used method for sytemic infection in a variety of infectious disease models in mice. This suggests that the CC003/Unc mice do not capture certain aspects of typhoid disease that occurs in humans, such as intestinal pathology, and bacterial shedding in faeces. Screening more CC strains to S. Typhi susceptibility might help identify specific CC strains that can capture the oral infection characteristics and intestinal pathology seen in humans.
In conclusion, the immunocompetent, S. Typhi permissive CC mouse model using CC003/Unc and CC053/ Unc presented here can provide an in vivo experimental system to evaluate novel preventive and therapeutic interventions. Additionally, genomic analyses of the CC mouse model can help us understand why responses to different ViPS conjugate vaccines vary among populations in disease-endemic countries 10 and why some individuals become chronic and asymptomatic carriers for spreading typhoid. This CC mouse model system can also enable the S. Typhi research community to identify putative S. Typhi virulence factor(s) and characterize their role in the progression of typhoid.   www.nature.com/scientificreports/ micro-isolator cages with free access to food and water and were maintained and bred in a specific pathogen-free facility at TJU. Three-week-old mice were considered young and 8-12-week-old mice were considered adult.

Methods
Infection. For mouse infections S. Typhi strain Ty2, a well-studied strain (obtained from Dr. Andreas Baumler) was grown to an OD 600 of ~ 1.0 in Luria Bertani (LB) broth containing 10 mM NaCl. Bacteria were washed twice in Dulbecco's phosphate-buffered saline (DPBS); and bacterial density was adjusted to 2 × 10 5 colony forming units (CFU)/ml. Mice were infected intraperitoneally (i.p.) with 2 × 10 4 CFU in 100 μl of DPBS. Because organs of the mononuclear phagocytic system, such as spleen and liver, are the major sites of replication of S. Typhi in humans, we assessed bacterial burden in these organs 6 days later to allow time for a detectable S. Typhi replication or host clearance. On day 6 post-infection, liver and spleen were collected and tissues were processed using a Minilys tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Bacterial burden in these tissue homogenates was measured by counting CFU on LB agar plates.
Histopathology analysis. Liver tissues obtained on day 6 post-infection were fixed in 10% buffered formalin and 4 μM paraffin-embedded sections were stained with hematoxylin and eosin. The specimen slides were scanned at 20 × magnification on Aperio CS2 Scanscope® (Leica Biosystems Inc.) followed by observer-blind histopathological analysis. Immunization. Two and a half μg of Vi Polysaccharide (ViPS; Lot 5 PDMI 158,299 obtained from the U.S. Food and Drug Administration, Silver Spring, MD) dissolved in 100 µl DPBS was used to immunize mice i.p. For whole bacterial immunization, mice were injected i.p. with 3 × 10 8 CFUs of heat-killed S. Typhi strain Ty2 in 100 µl DPBS 35 . The expression of ViPS is confirmed by serologically by slide agglutination test using a commercial Vi monoclonal antibody reagent (Statens Serum Institut diagnostica A/S, Denmark; Lot 188L-8). We also perform serum bactericidal assay using anti-S. Typhi human IgG (Lot R1, 2011; U.S. Food and Drug Administration, Silver Spring, MD) as described 35,52,63 . Blood samples were obtained 0-, 7-, 14-or 21-days following immunization and stored at − 20 °C.
Enzyme-linked immunosorbent assay (ELISA). ViPS-specific IgM and IgG were measured by coating 96-well microtiter plates (Nunc MaxiSorp™; Invitrogen, Carlsbad, CA) with 2 µg/ml of ViPS purified from S. Typhi clinical isolate C6524 64 in DPBS overnight at room temperature. All plates were washed and blocked with 2% Bovine serum albumin (BSA) in PBS pH 7.2 (blocking buffer) for 2 h at room temperature. Blood from immunized mice was diluted to 1:25 for IgG detection and 1:50 for IgM detection in blocking buffer; ViPSspecific mouse IgM and IgG levels were interpreted as ng/μl "equivalents" using normal mouse serum standards (Bethyl Laboratories, Montgomery, TX), as described previously 35 .
Serum bactericidal assay (SBA). SBA was performed as previously described 35 . In brief, log-phase cultures (OD 600 of 0.5 at 37 °C) of S. Typhi strain Ty2 were prepared in LB broth with 10 mM NaCl. Bacterial cells were washed in DPBS, and the bacterial cell density was adjusted to 1-3.5 × 10 4 CFU/ml in DPBS. Serum samples were heat-inactivated by incubating at 56 °C for 30 min prior to use in the assay. Ten microliters of S. Typhi cells in DPBS (100-350 CFU) were added to each well of a round-bottom polypropylene 96-well plate containing 50 μl of heat-inactivated serum in serial dilutions, 12.5 μl baby rabbit complement (Pel-Freeze, Rogers, AR), and 27.5 μl DPBS. Triplicate samples of each dilution were incubated for 120 min at 37 °C with gentle rocking, and 10 μl of this mixture was plated on LB agar plates for enumerating bacterial CFU. Serum bactericidal antibody titers were defined as the reciprocal of the highest dilution that produced > 50% killing in relation to control wells containing complement, but no mouse serum.
Statistical analysis. Data presented throughout depict pooled data from at least two independent experiments unless otherwise noted. Statistics were performed using the Prism 5 software program (GraphPad Software, Inc., La Jolla, CA). www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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